(1) Field of the Invention
The present invention relates to novel monoclonal antibodies against aflatoxin B.sub.1 and G.sub.1 and to a test kit and a method which uses these monoclonal antibodies to detect the presence of aflatoxin B.sub.1 and G.sub.1 in foods and other materials. In particular the present invention relates to monoclonal antibodies produced (1) by repeated introduction of aflatoxin B.sub.1 as a 1-position polypeptide conjugate into a murine over a period of time so that polyclonal antibodies are released into the blood serum of the murine and then (2) by the production of hybridomas from spleen cells of the murine which generate the monoclonal antibodies and which have substantially the cross-reactivity of the specific monoclonal antibody produced by hybridoma IVI-10108 to aflatoxin B.sub.1 and G.sub.1 and limited cross-reactivity to aflatoxins B.sub.2, G.sub.2 and M.sub.1.
(2) Prior Art
Aflatoxins are toxic metabolites produced by the fungal species Aspergillus flavus and Aspergillus parasiticus. The ability of aflatoxin B.sub.1 and its metabolites to act as potent carcinogens, mutagens and teratogens has been described (Butler, W. H. Mycotoxins p. 1-28 (1974); Chu, F. S., Adv. Appl. Microbiol. 22, 83 (1971)). Interest in development of rapid sensitive assays for detection of aflatoxins has been steadily increasing, since the compounds are known to occur naturally in peanuts, corn, milk, wheat, and animal rations (Butler, W. H. Mycotoxins p. 1-28 (1974)). Use of high performance liquid chromatography has been described for quantitation of aflatoxins (Gregory, J. F. and Manley, D. B., J. Assoc. Off. Anal. Chem. 65, 869 (1982); Stubblefield, R. D. and Shotwell, O. L., J. Assoc. Anal. Chem. 60. 4066 (1977)). Several drawbacks to using this procedure as a quick screening method include the high cost of the instrumentation, the need for extensive sample clean up, and only single samples may be analyzed at one time.
A number of immunoassays using polyclonal antiserum have been described for detection of aflatoxin B.sub.1 (Chu, F. S. and Ueno, I. Appl. Environ. Microbiol. 33, 1125 (1977); El-Nakib, O., Pestka, J. J. and Chu, F. S., J. Assoc. Off. Anal. Chem. 64, 1077 (1981); Langone, J. L. and Van Vunakis, H., J. Natl. Cancer Inst. 56, 591 (1976); Lawellin, D. W., Grant, D. W. and Joyce, B. K., Appl. Environ. Microbiol. 34, 94 (1977); Pestka, J. J., Gaur, P. K. and Chu, F. S., Appl. Environ. Microbiol. 40, 1027 (1980); and Pestka, J. J. and Chu, F. S., J. Fd. Prot. 47, 305 (1984)). The advantages offered by these assays include a reduction in assay time, simplified extraction procedures, an increase in assay sensitivity, and the ability to routinely screen large numbers of samples.
In an attempt to compensate for the limitations encountered when using conventional polyclonal antisera assay systems (availability, potential antibody variation from bleeding to bleeding, and problems with large scale production of antibody), hybridoma cell lines, which secrete monoclonal antibodies to aflatoxin B.sub.1 (or aflatoxin B.sub.1 -DNA adducts), have been produced (Candlish, A. A. G., Stimson, W. H. and Smith, J. E., Letters in Appl. Microbiol. 1, 57 (1985); Groopman, J. D., Haugen, A., Goodrich, G. R., Wogan, G. N. and Harris, C. C., Cancer Res. 42, 3120 (1982); Groopman, J. D. Trudel, L. J., Donahue, P. R., Marshak-Rothstein, A. and Wogan, G. N., Proc. Natl. Acad. Sci. U.S.A. 81, 7728 (1984); Haugen, A., Groopman, J. D., Hsu, I. C., Goodrich, G. R., Wogan, G. N. and Harris, C. C., Proc. Natl. Acad. Sci. U.S.A. 18, 4124 (1981)). The potential exists for the availability of an infinite supply of homogeneous antibody which can be mass produced. In a rapid assay (e.g. ten minutes) there is too little cross-reactivity with G.sub.1 using these prior art monoclonal antibodies.
Thus monoclonal antibodies which recognize aflatoxin B.sub.1, B.sub.2 and M.sub.1 are well known as shown by Candlish, A. A. G., Stimson, W. H. and Smith, J. E., Letters in Appl. Microbiol. 1, 57 (1985); Groopman, J. D., Trudel, L. J., Donahue, P. R., Marshak-Rothstein, A. and Wogan, G. N., Proc. Natl. Acad. Sci. USA 81, 7728 (1984). There is a need for monoclonal antibodies which react significantly with only B.sub.1 and G.sub.1. Aflatoxin G.sub.1, is a major source of aflatoxin contamination in peanuts.
It would be advantageous to use monoclonal antibodies for conducting the immunoassays for aflatoxin B.sub.1 and G.sub.1. Hybridomas produce unlimited amounts of highly uniform monoclonal antibodies. The monoclonal antibodies can then be used in the development of a colorimetric commercial assay systems for the mycotoxins, such as Enzyme Linked Immunosorbent Assay (ELISA) or fluorescent antibody tests.